The present invention relates to mechanically assisted methods, and apparatus therefore, to remove cryoprotectant from suspensions of cells in need of cryoprotection.
Cryopreservation is a procedure for preparation of a suspension of cells, or a group of cells such as an embryo, for storage. The procedure normally incorporates adding cryoprotectants to the cells to be preserved, cooling of the suspended cells, long-term storage of the cell suspension at temperatures below about xe2x88x9280xc2x0 C., warming of the cells to normal cell temperatures, and removal of cryoprotectant from the cells. Cryopreservation of sperm or other cells from common mammals is a deceptively simple-appearing process which succeeds despite certain serious obstacles. This success depends on the use of one or more cryoprotectants in the context of certain procedural parameters.
Overall cryopreservation procedures thus generally include: preparation of a suspension of cells for low-temperature storage by incorporation of cryoprotectants, and placement of individual units into vials or xe2x80x9cstrawsxe2x80x9d; cooling (sometimes called xe2x80x9cfreezingxe2x80x9d) at an appropriate rate; long-term storage of the suspension of cells at a temperature lower than xe2x88x9280xc2x0 C. and often between xe2x88x92180xc2x0 C. and xe2x88x92196xc2x0 C.; distribution at low temperature to intermediaries or users; warming (sometimes called xe2x80x9cthawingxe2x80x9d) at an appropriate rate to the normal cellular temperature; and controlled removal of cryoprotectant plus any other medium or other adjustments needed to render the cells ready for in vivo use. The goal is not just to keep cells alive (viable), but to optimize retention of all cellular attributes such as normal life span, oxygen-carrying potential (especially in the case of erythrocytes) and fertilizing potential (in the case of spermatozoa or oocytes), for which the cells are being preserved in the first place.
Notwithstanding cell type, species of origin or the various protocols used, prior art cryopreservation protocols traditionally result in about 30% mortality (or worse) of cells being preserved. Many cells traditionally did not survive the cooling and rewarming, and those which did suffered further damage during removal of the intracellular cryoprotectant. Damage can result from any or all of improper rates of temperature changes during cooling and rewarming, formation of ice crystals, reduction in temperature per se, toxicity due to high concentrations of solutes within and around the cells, the nature and concentration of the cryoprotectant(s) used, rates of addition and removal of cryoprotectants from within the cells, and other lesser known but empirically evident factors.
A cryoprotectant is a molecule which allows a substantial percentage of cells to survive a freeze-thaw cycle and to retain normal cell function. Cryoprotectants which pass through the cell plasma membrane, and which thus act both intracellularly and extracellularly, are termed penetrating cryoprotectants. Non-penetrating cryoprotectants act only extracellularly. Glycerol is the most effective penetrating cryoprotectant for certain types of cells and for sperm from most species. Glycerol is of low toxicity, relative to the alternative penetrating cryoprotectants such as ethylene glycol, propylene glycol and dimethylsulfoxide. All penetrating cryoprotectants pass through a cellular membrane at a rate slower than water does, and each of these rates is itself temperature dependent. Non-penetrating cryoprotectants include proteins (such as milk or egg proteins used with mammalian sperm); sugars such as lactose, fructose, raffinose or trehalose; synthetic polymers such as methyl cellulose; and amide compounds. Most penetrating cryoprotectants, such as glycerol, serve as a solute (and cause osmotic flow of water) and a solvent (to dissolve salts and sugars) miscible with water. All non-penetrating cryoprotectants are solutes or colloids, and cannot themselves also serve as solvents. Both water and glycerol, as well as other solvents, pass through the membrane of cells and eventually equilibrate at the same concentration in all internal structures, so that the intracellular and extracellular concentrations are the same.
The solute role of a penetrating cryoprotectant is believed to cause damage due to the induced osmotic flow of water. The solvent role is beneficial, however, because the penetrating cryoprotectants such as glycerol have a freezing point much lower than that of water. In the presence of glycerol, the portion of the solvent mixture remaining unfrozen at any given temperature is greater than if water were the only solvent. Hence, at any given temperature there is more xe2x80x9cspacexe2x80x9d for the cells in channels of unfrozen solvent and a lower concentration of solutes (the same amount of solutes is contained in more liquid). This phenomenon occurs both inside and outside the cells. Further, the presence of glycerol probably reduces formation of micro-fractures in the ice and this, in turn, minimizes damage to cells. Non-penetrating cryoprotectants, such as sugars and lipoproteins, typically are present in relatively high concentrations. They typically act by modifying the plasma membrane, so that it is more resistant to temperature-induced damage, or simply by acting as a solute to lower the freezing point of the solute/solvent combination.
Conventional procedures for preservation of many cells involve abrupt addition of a penetrating cryoprotectant, such as glycerol, to a cell suspension despite both long-standing and recent warnings that penetrating cryoprotectant should be added slowly. Similarly, the benefit of slow removal of penetrating cryoprotectant from cells is well known. Damage associated with the rapid addition or removal of a penetrating cryoprotectant is a direct consequence of extreme changes in cell volume, resulting from rapid movement of water, and formation of irreversible xe2x80x9ctearsxe2x80x9d in the plasma membrane. Slow removal of cryoprotectants from within thawed cells generally has not been used, either because of ignorance or lack of a convenient approach to achieve it.
Depending on the number of cells required for a functional xe2x80x9cunitxe2x80x9d after thawing, cells traditionally are packaged as individual units using glass ampules, plastic vials, plastic straws, or appropriately sized plastic bags. These packages all require removal of the cell suspension from the primary container before slow removal of cryoprotectant. Alternatively, technology disclosed by Hammerstedt et al. (U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870, both incorporated herein by reference) allows slow removal of cryoprotectant while the cells remain within the primary container, via open pores in a special membrane formed to provide a primary container which allows exchange of fluid across the membrane. Although efficacious, because this approach is diffusion-limited the process can require up to two hours to reduce the concentration of cryoprotectant within the cells to a desired level.
Apart from the widely practiced stepwise dilution used to address this problem, slow removal of cryoprotectant from cells in a suspension can be accomplished by placing the suspension, after thawing, into a conventional dialysis membrane and suspending the dialysis unit in a large volume of a salts solution. Alternatively, special plugged-pore containers, such as those disclosed in the U.S. patents incorporated by reference above, can be processed after thawing in a manner to open the pores of the membrane-container and to allow movement of molecules across the membrane. In both cases, movement of cryoprotectant or water through the membrane of the primary container is via diffusion and xe2x80x9cdownxe2x80x9d the concentration gradient (i.e., away from the locus of highest concentration). Such diffusion-based processes effectively limit the rates at which composition of the medium immediately surrounding the cells is altered as water and cryoprotectant diffuse, in and out respectively, across the primary container membrane. Consequently, this limits rates of movement of water and cryoprotectant across the membranes of the cells to flux degrees which are not damaging to the cells. Reliance on simple diffusion through the membrane container requires a commercially unacceptably long 1-2 hour time period.
The alternative assisted cryoprotectant removal method uses continuous flow centrifugation, such as is discussed in U.S. Pat. No. 4,221,322. This requires transfers from the original cryopreservation packaging, thus affording opportunity for contamination and requiring excessive processing.
A need thus remains for a method for introducing and removing one or more cryoprotectants, to and from a cell or group of cells in need of cryopreservation, in which the cellular damage, potential contamination and/or long processing times of the prior art are avoided.
In order to meet this need, the present invention embodies a process and apparatus in which cryoprotectant in cryopreserved samples is removed with the use of turbulence and convective-dispersion around the cells within the primary cryopreservation container, applied via the combined action of gravity and pulsatile transmembrane flow of buffer through the pores of primary containers fabricated from appropriate membrane material such as that disclosed in U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870, as incorporated herein by reference above. By using mechanical forces to aid the process of cryoprotectant removal, the benefits of slow cryoprotectant removal without transferring out of the original primary container are retained, while the actual removal time is markedly decreased and sterility maintained.